A structural model for the ternary cleavable complex formed between human topoisomerase I, DNA, and camptothecin.

Using the X-ray crystal structure of the human topoisomerase I (TOP1)-DNA cleavable complex, we have developed a general model for the ternary drug-DNA-TOP1 cleavable complex formed with camptothecin (CPT) and its analogues. This model has the drug intercalated between the -1 and +1 base pairs, with the E-ring pointing into the minor groove and the A-ring directed toward the major groove. The ternary complex is stabilized by an array of hydrogen bonding and hydrophobic interactions between the drug and both the enzyme and the DNA. Significantly, the proposed model is consistent with the current body of experimental mutation, cross-linking, and structure-activity data. In addition, the model reveals potential sites of interaction that can provide a rational basis for the design of next generation compounds as well as for de novo drug design.

Calorimetry of nucleic acids.

This unit describes the application of differential scanning and isothermal titration calorimetry (DSC and ITC) to the study of the thermodynamics of nucleic acid structure. DSC is used to study order-disorder transitions. A single DSC profile provides a wealth of thermodynamic and extrathermodynamic information: transition enthalpy, entropy, free energy, heat capacity, the state of the transition (two-state vs. multistate), and the size of the cooperative unit. ITC is used to study hybridization of nucleic acids at constant temperature. Results can be used to determine the stoichiometry of the association reaction, the enthalpy of association, the equilibrium association constant, and the free energy and entropy of association. A thorough discussion is presented of the details required to obtain meaningful results, as well as relevant methods for analyzing the data produced.

Site-specific topoisomerase I-mediated DNA cleavage induced by nogalamycin: a potential role of ligand-induced DNA bending at a distal site.

Many DNA binding ligands (e.g., nogalamycin, actinomycin D, terbenzimidazoles, indolocarbazoles, nitidine, and coralyne) and various types of DNA lesions (e.g., UV dimers, DNA mismatches, and abasic sites) are known to stimulate topoisomerase I-mediated DNA cleavage. However, the mechanism(s) by which these covalent and noncovalent DNA interactions stimulate topoisomerase I-mediated DNA cleavage remains unclear. Using nogalamycin as a model, we have studied the mechanism of ligand-induced topoisomerase I-mediated DNA cleavage. We show by both mutational and DNA footprinting analyses that the binding of nogalamycin to an upstream site (from position -6 to -3) can induce highly specific topoisomerase I-mediated DNA cleavage. Substitution of this nogalamycin binding site with a DNA bending sequence (A(5)) stimulated topoisomerase I-mediated DNA at the same site in the absence of nogalamycin. Replacement of the A(5) sequence with a disrupted DNA bending sequence (A(2)TA(2)) significantly reduced the level of topoisomerase I-mediated DNA cleavage. These results, together with the known DNA bending property of nogalamycin, suggest that the nogalamycin-DNA complex may provide a DNA structural bend to stimulate topoisomerase I-mediated DNA cleavage.

2"-Substituted 5-phenylterbenzimidazoles as topoisomerase I poisons.

5-Phenylterbenzimidazole (1) is active as a topoisomerase I poison (topo I) and is cytotoxic to human tumor cells. No cross-resistance was observed for 1 when it was evaluated against the camptothecin-resistant cell line, CPT-K5. Derivatives of 1 substituted at the 2"-position, however, did exhibit cross-resistance to this cell line. The basis for the resistance of this cell line towards CPT is that it possesses a mutant form of topo I. These results suggest that substituents at the 2"-position may be in proximity to the wild-type enzyme. Therefore, we hypothesized that terbenzimidazoles with 2"-substituents could be capable of interacting with the enzyme and thereby influence activity within this class of topo I poisons. 5-Phenylterbenzimidazoles with a hydroxy, hydroxymethyl, mercapto, amino, N-benzoylaminomethyl, chloro, and trifluoromethyl group at the 2"-position were synthesized. In addition, several 2"-ethyl-5-phenylterbenzimidazoles were prepared containing either a methoxy, hydroxy, amino, or N-acetylamino group at the 2-position of the ethyl side-chain. These 2"-substituted 5-phenylterbenzimidazoles were evaluated as topo I poisons and for cytotoxic activity. The presence of a strong electron-withdrawing group at the 2"-position, such as a chloro or trifluoromethyl group, did enhance both topo I poisoning activity and cytotoxicity. Studies on the relative DNA binding affinity of 1 to its 2"-amino and 2"-trifluoromethyl derivatives did exhibit a correlation with their relative differences in biological activity.

Human topoisomerase I poisoning by protoberberines: potential roles for both drug-DNA and drug-enzyme interactions.

Protoberberines represent a structural class of organic cations that induce topoisomerase I-mediated DNA cleavage, a behavior termed topoisomerase I poisoning. We have employed a broad range of biophysical, biochemical, and computer modeling techniques to characterize and cross-correlate the DNA-binding and topoisomerase poisoning properties of four protoberberine analogues that differ with respect to the substituents on their A- and/or D-rings. Our data reveal the following significant features: (i) The binding of the four protoberberines unwinds duplex DNA by approximately 11 degrees, an observation consistent with an intercalative mode of interaction. (ii) Enthalpically favorable interactions, such as stacking interactions between the intercalated ligand and the neighboring base pairs, provide <50% of the thermodynamic driving force for the complexation of the protoberberines to duplex DNA. Computer modeling studies on protoberberine-DNA complexes suggest that only rings C and D intercalate into the host DNA helix, while rings A and B protrude out of the helix interior into the minor groove. (iii) All four protoberberine analogues are topoisomerase I-specific poisons, exhibiting little or no topoisomerase II poisoning activity. (iv) Modifications of the D-ring influence both DNA binding and topoisomerase I poisoning properties. Specifically, transference of a methoxy substituent from the 11- to the 9-position diminishes both DNA binding affinity and topoisomerase I poisoning activity, an observation suggesting that DNA binding is important in the poisoning of topoisomerase I by protoberberines. (v) Modifications of the A-ring have a negligible impact on DNA binding affinity, while exerting a profound influence on topoisomerase I poisoning activity. Specifically, protoberberine analogues containing either 2,3-dimethoxy; 3,4-dimethoxy; or 3, 4-methylenedioxy substituents all bind DNA with a similar affinity. By contrast, these analogues exhibit markedly different topoisomerase I poisoning activities, with these activities following the hierarchy: 3,4-methylenedioxy > 2,3-dimethoxy >> 3, 4-dimethoxy. These differences in topoisomerase I poisoning activity may reflect the differing abilities of the analogues to interact with specific functionalities on the enzyme, thereby stabilizing the enzyme in its cleavable state. In the aggregate, our results are consistent with a mechanistic model in which both ligand-DNA and ligand-enzyme interactions are important for the poisoning of topoisomerase I by protoberberines, with the DNA-directed interactions involving ring D and the enzyme-directed interactions involving ring A. It is reasonable to suggest that the poisoning of topoisomerase I by a broad range of other naturally occurring and synthetic ligands may entail a similar mechanism.

Aminoglycoside binding in the major groove of duplex RNA: the thermodynamic and electrostatic forces that govern recognition.

We use a combination of spectroscopic, calorimetric, viscometric and computer modeling techniques to characterize the binding of the aminoglycoside antibiotic, tobramycin, to the polymeric RNA duplex, poly(rI).poly(rC), which exhibits the characteristic A-type conformation that is conserved among natural and synthetic double-helical RNA sequences. Our results reveal the following significant features: (i) CD-detected binding of tobramycin to poly(rI).poly(rC) reveals an apparent site size of four base-pairs per bound drug molecule; (ii) tobramycin binding enhances the thermal stability of the host poly(rI).poly(rC) duplex, the extent of which decreases upon increasing in Na(+) concentration and/or pH conditions; (iii) the enthalpy of tobramycin- poly(rI).poly(rC) complexation increases with increasing pH conditions, an observation consistent with binding-induced protonation of one or more drug amino groups; (iv) the affinity of tobramycin for poly(rI).poly(rC) is sensitive to both pH and Na(+) concentration, with increases in pH and/or Na(+) concentration resulting in a concomitant reduction in binding affinity. The salt dependence of the tobramycin binding affinity reveals that the drug binds to the host RNA duplex as trication. (v) The thermodynamic driving force for tobramycin- poly(rI).poly(rC) complexation depends on pH conditions. Specifically, at pH< or =6.0, tobramycin binding is entropy driven, but is enthalpy driven at pH > 6.0. (vi) Viscometric data reveal non-intercalative binding properties when tobramycin complexes with poly(rI).poly(rC), consistent with a major groove-directed mode of binding. These data also are consistent with a binding-induced reduction in the apparent molecular length of the host RNA duplex. (vii) Computer modeling studies reveal a tobramycin-poly(rI). poly(rC) complex in which the drug fits snugly at the base of the RNA major groove and is stabilized, at least in part, by an array of hydrogen bonding interactions with both base and backbone atoms of the host RNA. These studies also demonstrate an inability of tobramycin to form a stable low-energy complex with the minor groove of the poly(rI).poly(rC) duplex. In the aggregate, our results suggest that tobramycin-RNA recognition is dictated and controlled by a broad range of factors that include electrostatic interactions, hydrogen bonding interactions, drug protonation reactions, and binding-induced alterations in the structure of the host RNA. These modulatory effects on tobramycin-RNA complexation are discussed in terms of their potential importance for the selective recognition of specific RNA structural motifs, such as asymmetric internal loops or hairpin loop-stem junctions, by aminoglycoside antibiotics and their derivatives.

Heterocyclic bibenzimidazole derivatives as topoisomerase I inhibitors.

A series of 2'-heterocyclic derivatives of 5-phenyl-2,5'-1H-bibenzimidazoles were evaluated for topoisomerase I poisoning activity and cytotoxicity. Topo I poisoning activity was associated with 2'-derivatives that possessed a hydrogen atom capable of hydrogen bond formation, suggesting that the interatomic distances between such hydrogen atoms and the heteroatoms on the adjacent benzimidazole influence activity.

DNA sequence context modulates the impact of a cisplatin 1,2-d(GpG) intrastrand cross-link on the conformational and thermodynamic properties of duplex DNA.

The anticancer activity of cisplatin derives from its ability to bind and cross-link DNA, with the major adduct being the 1,2-d(GpG) intrastrand cross-link. Here, the consequences of this adduct on the conformation, thermal stability, and energetics of duplex DNA are assessed, and the modulation of these parameters by the sequence context of the adduct is evaluated. The properties of a family of 15-mer DNA duplexes containing a single 1,2-d(GpG) cis-¿Pt(NH(3))(2)¿(2+) intrastrand cross-link are probed in different sequence contexts where the flanking base-pairs are systematically varied from T.A to C.G to A.T. By using a combination of spectroscopic and calorimetric techniques, the structural, thermal, and thermodynamic properties of each duplex, both with and without the cross-link, are characterized. Circular dichroism spectroscopic data reveal that the cross-link alters the structure of the host duplex in a manner consistent with a shift from a B-like to an A-like conformation. Thermal denaturation data reveal that the cross-link induces substantial thermal and thermodynamic destabilization of the host duplex. Significantly, the magnitudes of these cross-link-induced effects on duplex structure, thermal stability, and energetics are influenced by the bases that flank the adduct. The presence of flanking A.T base-pairs, relative to T.A or C.G base-pairs, enhances the extent of cross-link-induced alteration to an A-like conformation and dampens the extent of cross-link-induced duplex destabilization. These results are discussed in terms of available structural data, and in terms of the selective recognition of cisplatin-DNA adducts by HMG-domain proteins.

The thermodynamics of polyamide-DNA recognition: hairpin polyamide binding in the minor groove of duplex DNA.

Crescent-shaped synthetic ligands containing aromatic amino acids have been designed for specific recognition of predetermined DNA sequences in the minor groove of DNA. Simple rules have been developed that relate the side-by-side pairings of Imidazole (Im) and Pyrrole (Py) amino acids to their predicted target DNA sequences. We report here thermodynamic characterization of the DNA-binding properties of the six-ring hairpin polyamide, ImImPy-gamma-PyPyPy-beta-Dp (where gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide). Our data reveal that, at 20 degrees C, this ligand binds with a relatively modest 1.8-fold preference for the designated match site, 5'-TGGTA-3', over the single base pair mismatch site, 5'-TGTTA-3'. By contrast, we find that the ligand exhibits a 102-fold greater affinity for its designated match site relative to the double base pair mismatch site, 5'-TATTA-3'. These results demonstrate that the energetic cost of binding to a double mismatch site is not necessarily equal to twice the energetic cost of binding to a single mismatch site. Our calorimetrically measured binding enthalpies and calculated entropy data at 20 degrees C reveal the ligand sequence specificity to be enthalpic in origin. We have compared the DNA-binding properties of ImImPy-gamma-PyPyPy-beta-Dp with the hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (an Im --> Py "mutant"). Our data reveal that both ligands exhibit high affinities for their designated match sites, consistent with the Dervan pairing rules. Our data also reveal that, relative to their corresponding single mismatch sites, ImImPy-gamma-PyPyPy-beta-Dp is less selective than ImPyPy-gamma-PyPyPy-beta-Dp for its designated match site. This result suggests, at least in this case, that enhanced binding affinity can be accompanied by some loss in sequence specificity. Such systematic comparative studies allow us to begin to establish the thermodynamic database required for the rational design of synthetic polyamides with predictable DNA-binding affinities and specificities.

DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.

When mammalian cell cultures or mice are exposed to ionizing radiation in survivable or lethal amounts, novel mass components are found in the histone H2A region of two-dimensional gels. Collectively referred to as gamma, these components are formed in vivo by several procedures that introduce double-stranded breaks into DNA. gamma-Components, which appeared to be the only major novel components detected by mass or 32PO4 incorporation on acetic acid-urea-Triton X-100-acetic acid-urea-cetyltrimethylammonium bromide or SDS-acetic acid-urea-cetyltrimethylammonium bromide gels after exposure of cells to ionizing radiation, are shown to be histone H2AX species that have been phosphorylated specifically at serine 139. gamma-H2AX appears rapidly after exposure of cell cultures to ionizing radiation; half-maximal amounts are reached by 1 min and maximal amounts by 10 min. At the maximum, approximately 1% of the H2AX becomes gamma-phosphorylated per gray of ionizing radiation, a finding that indicates that 35 DNA double-stranded breaks, the number introduced by each gray into the 6 x 10(9) base pairs of a mammalian G1 genome, leads to the gamma-phosphorylation of H2AX distributed over 1% of the chromatin. Thus, about 0.03% of the chromatin appears to be involved per DNA double-stranded break. This value, which corresponds to about 2 x 10(6) base pairs of DNA per double-stranded break, indicates that large amounts of chromatin are involved with each DNA double-stranded break. Thus, gamma-H2AX formation is a rapid and sensitive cellular response to the presence of DNA double-stranded breaks, a response that may provide insight into higher order chromatin structures.

DNA minor groove binding-directed poisoning of human DNA topoisomerase I by terbenzimidazoles.

We have employed a broad range of spectroscopic, calorimetric, DNA cleavage, and DNA winding/unwinding measurements to characterize the DNA binding and topoisomerase I (TOP1) poisoning properties of three terbenzimidazole analogues, 5-phenylterbenzimidazole (5PTB), terbenzimidazole (TB), and 5-(naphthyl[2,3-d]imidazo-2-yl)bibenzimidazole (5NIBB), which differ with respect to the substitutions at their C5 and/or C6 positions. Our results reveal the following significant features. (i) The overall extent to which the three terbenzimidazole analogues poison human TOP1 follows the hierarchy 5PTB > TB >> 5NIBB. (ii) The impact of the three terbenzimidazole analogues on the superhelical state of plasmid DNA depends on the [total ligand] to [base pair] ratio (rbp), having no effect on DNA superhelicity at rbp ratios < or = 0.1, while weakly unwinding DNA at rbp ratios > 0.1. This weak DNA unwinding activity exhibited by the three terbenzimidazoles does not appear to be correlated with the abilities of these compounds to poison TOP1. (iii) Upon complexation with both poly(dA).poly(dT) and salmon testes DNA, the three terbenzimidazole analogues exhibit flow linear dichroism properties characteristic of a minor groove-directed mode of binding to these host DNA duplexes. (iv) The apparent minor groove binding affinities of the three terbenzimidazole analogues for the d(GA4T4C)2 duplex follow a qualitatively similar hierarchy to that noted above for ligand-induced poisoning of human TOP1-namely, 5PTB > TB > 5NIBB. In the aggregate, our results suggest that DNA minor groove binding, but not DNA unwinding, is important in the poisoning of TOP1 by terbenzimidazoles.

Minor groove-directed and intercalative ligand-DNA interactions in the poisoning of human DNA topoisomerase I by protoberberine analogs.

Spectroscopic, calorimetric, DNA cleavage, electrophoretic, and computer modeling techniques have been employed to characterize the DNA binding and topoisomerase poisoning properties of three protoberberine analogs, 8-desmethylcoralyne (DMC), 5,6-dihydro-8-desmethylcoralyne (DHDMC), and palmatine, which differ in the chemical structures of their B- and/or D-rings. DNA topoisomerase-mediated cleavage assays revealed that these compounds were unable to poison mammalian type II topoisomerase. By contrast, the three protoberberine analogs poisoned human topoisomerase I according to the following hierarchy: DHDMC > DMC > palmatine. DNA binding by all three protoberberine analogs induced negative flow linear dichroism signals as well as unwinding of the host duplex. These two observations are consistent with an intercalative mode of protoberberine binding to duplex DNA. However, a comparison of the DNA binding properties for DMC and DHDMC, which differ only by the state of saturation at the 5,6 positions of the B-ring, revealed that the protoberberine analogs do not "behave" like classic DNA intercalators. Specifically, saturation of the 5-6 double bond in the B-ring of DMC, thereby converting it to the DHDMC molecule, was associated with enhanced DNA unwinding as well as a reversal of DNA binding preference from a DNA duplex with an inaccessible or occluded minor groove {poly[d(G-C)]2} to DNA duplexes with accessible or unobstructed minor grooves {poly[d(A-T)]2 and poly[d(I-C)]2}. In addition, a comparison of the DNA binding properties for DHDMC and palmatine revealed that transferring the 11-methoxy moiety on the D-ring of DHDMC to the 9 position, thereby converting it to palmatine, was associated with a reduction in binding affinity for both duplexes with unobstructed minor grooves as well as for duplexes with occluded minor grooves. These DNA binding properties are consistent with a "mixed-mode" DNA binding model for protoberberines in which a portion of the ligand molecule intercalates into the double helix, while the nonintercalated portion of the ligand molecule protrudes into the minor groove of the host duplex, where it is thereby available for interactions with atoms lining the floor and/or walls of the minor groove. Furthermore, saturation at the 5,6 positions of the B-ring, which causes the A-ring to be tilted relative to the plane formed by rings C and D, appears to stabilize the interaction between the host duplex and the minor groove-directed portion of the protoberberine ligand. Computer modeling studies on the DHDMC-poly[d(A-T)]2 complex suggest that this interaction may involve van der Waals contacts between the ligand A-ring and backbone sugar atoms lining the minor groove of the host duplex. The hierarchy of topoisomerase I poisoning noted above suggests that this minor groove-directed interaction may play an important role in topoisomerase I poisoning by protoberberine analogs. In the aggregate, our results presented here, coupled with the recent demonstration of topoisomerase I poisoning by minor groove-binding terbenzimidazoles [Sun, Q., Gatto, B., Yu, C., Liu, A. , Liu, L. F., & LaVoie, E. J. (1995) J. Med. Chem. 38, 3638-3644], suggest that minor groove-directed ligand-DNA interactions may be of general importance in the poisoning of topoisomerase I.

Differential poisoning of topoisomerases by menogaril and nogalamycin dictated by the minor groove-binding nogalose sugar.

The effect of DNA binding on poisoning of human DNA TOP1 has been studied using a pair of related anthracyclines which differ only by a nogalose sugar ring. We show that the nogalose sugar ring of nogalamycin, which binds to the minor groove of DNA, plays an important role in affecting topoisomerase-specific poisoning. Using purified mammalian topoisomerases, menogaril is shown to poison topoisomerase II but not topoisomerase I. By contrast, nogalamycin poisons topoisomerase I but not topoisomerase II. Consistent with the biochemical studies, CEM/VM-1 cells which express drug-resistant TOP2alpha are cross-resistant to menogaril but not nogalamycin. The mechanism by which nogalamycin poisons topoisomerase I has been studied by analyzing a major topoisomerase I-mediated DNA cleavage site induced by nogalamycin. This site is mapped to a sequence embedded in an AT-rich region with four scattered GC base pairs (bps) (at -10, -6, +2, and +12 positions). GC bps embedded in AT-rich regions are known to be essential for nogalamycin binding. Surprisingly, DNase I footprinting analysis of nogalamycin-DNA complexes has revealed a drug-free region from -2 to +9 encompassing the major cleavage site. Our results suggest that nogalamycin, in contrast to camptothecin, may stimulate TOP1 cleavage by binding to a site(s) distal to the site of cleavage.

Modulation of nucleic acid structure by ligand binding: induction of a DNA.RNA.DNA hybrid triplex by DAPI intercalation.

The aromatic diamidine, DAPI (4',6-diamidino-2-phenylindole), is used as an important biological and cytological tool since it forms highly fluorescent complexes with nucleic acid duplexes via minor groove-directed/intercalative modes of interaction. In this study, we find that DAPI binding can induce the formation of an RNA-DNA hybrid triplex that would not otherwise form. More specifically, through application of a broad range of spectroscopic, viscometric, and molecular modeling techniques, we demonstrate that DAPI intercalation induces the formation of the poly(dT).poly(rA).poly(dT) hybrid triple helix, a structure which does not form in the absence of the ligand. Using UV mixing studies, we demonstrate that, in the presence of DAPI, the poly(rA).poly(dT) duplex and the poly(dT) single strand form a 1:1 complex (a triplex) that does not form in the absence of DAPI. Through temperature-dependent absorbance measurements, we show that the poly(dT).poly(rA).poly(dT) triplex melts via two distinct transitions: initial conversion of the triplex to the duplex state, with the DAPI remaining bound, followed by denaturation of the duplex-DAPI complex to its component single strands and free DAPI. Using optical melting profiles, we show that DAPI binding enhances the thermal stability of the poly(dT).poly(rA).poly(dT) triplex, an observation consistent with the preferential binding of the ligand to the triplex versus the duplex and single-stranded states. Our differential scanning calorimetric measurements reveal melting of the DAPI-saturated poly(dT).poly(rA).poly(dT) triplex to be associated with a lower enthalpy but greater cooperativity than melting of the corresponding DAPI-saturated poly(rA).poly(dT) duplex. Our flow linear dichroism and viscometric data are consistent with an intercalative mode of binding when DAPI interacts with both the poly(dT).poly(rA).poly(dT) triplex and the poly(rA).poly(dT) duplex. Finally, computer modeling studies suggest that a combination of both stacking and electrostatic interactions between the intercalated ligand and the host nucleic acid play important roles in the DAPI-induced stabilization of the poly(dT).poly(rA).poly(dT) triplex. In the aggregate, our results demonstrate that ligand binding can be used to induce the formation of triplex structures that do not form in the absence of the ligand. This triplex-inducing capacity has potentially important implications in the design of novel antisense, antigene, antiviral, and diagnostic strategies.

A terbenzimidazole that preferentially binds and conformationally alters structurally distinct DNA duplex domains: a potential mechanism for topoisomerase I poisoning.

The terbenzimidazoles are a class of synthetic ligands that poison the human topoisomerase I (TOP1) enzyme and promote cancer cell death. It has been proposed that drugs of this class act as TOP1 poisons by binding to the minor groove of the DNA substrate of TOP1 and altering its structure in a manner that results in enzyme-mediated DNA cleavage. To test this hypothesis, we characterize and compare the binding properties of a 5-phenylterbenzimidazole derivative (5PTB) to the d(GA4T4C)2 and d(GT4A4C)2 duplexes. The d(GA4T4C)2 duplex contains an uninterrupted 8-bp A.T domain, which, on the basis of x-ray crystallographic data, should induce a highly hydrated "A-tract" conformation. This duplex also exhibits anomalously slow migration in a polyacrylamide gel, a feature characteristic of a noncanonical global conformational state frequently described as "bent." By contrast, the d(GT4A4C)2 duplex contains two 4-bp A.T tracts separated by a TpA dinucleotide step, which should induce a less hydrated "B-like" conformation. This duplex also migrates normally in a polyacrylamide gel, a feature further characteristic of a global, canonical B-form duplex. Our data reveal that, at 20 degrees C, 5PTB exhibits an approximately 2. 3 kcal/mol greater affinity for the d(GA4T4C)2 duplex than for the d(GT4A4C)2 duplex. Significantly, we find this sequence/conformational binding specificity of 5PTB to be entropic in origin, an observation consistent with a greater degree of drug binding-induced dehydration of the more solvated d(GA4T4C)2 duplex. By contrast with the differential duplex affinity exhibited by 5PTB, netropsin and 4',6-diamidino-2-phenylindole (DAPI), two AT-specific minor groove binding ligands that are inactive as human TOP1 poisons, bind to both duplexes with similar affinities. The electrophoretic behaviors of the ligand-free and ligand-bound duplexes are consistent with 5PTB-induced bending and/or unwinding of both duplexes, which, for the d(GA4T4C)2 duplex, is synergistic with the endogenous sequence-directed electrophoretic properties of the ligand-free duplex state. By contrast, the binding to either duplex of netropsin or DAPI induces little or no change in the electrophoretic mobilities of the duplexes. Our results demonstrate that the TOP1 poison 5PTB binds differentially to and alters the structures of the two duplexes, in contrast to netropsin and DAPI, which bind with similar affinities to the two duplexes and do not significantly alter their structures. These results are consistent with a mechanism for TOP1 poisoning in which drugs such as 5PTB differentially target conformationally distinct DNA sites and induce structural changes that promote enzyme-mediated DNA cleavage.

Increasing the distance between the snRNA promoter and the 3' box decreases the efficiency of snRNA 3'-end formation.

Chimeric genes which contained the mouse U1b snRNA promoter, portions of the histone H2a or globin coding regions and the U1b 3'-end followed by a histone 3'-end were constructed. The distance between the U1 promoter and the U1 3' box was varied between 146 and 670 nt. The chimeric genes were introduced into CHO cells by stable transfection or into Xenopus oocytes by microinjection. The efficiency of utilization of the U1 3' box, as measured by the relative amounts of transcripts that ended at the U1 3' box and the histone 3'-end, was dependent on the distance between the promoter and 3'-end box. U1 3'-ends were formed with >90% efficiency on transcripts shorter than 200 nt, with 50-70% efficiency on transcripts of 280-400 nt and with only 10-20% efficiency on transcripts >500 nt. Essentially identical results were obtained after stable transfection of CHO cells or after injecting the genes into Xenopus oocytes. The distance between the U1 promoter and the U1 3' box must be <280 nt for efficient transcription termination at the U1 3' box, regardless of the sequence transcribed.

Binding of a hairpin polyamide in the minor groove of DNA: sequence-specific enthalpic discrimination.

Hairpin polyamides are synthetic ligands for sequence-specific recognition in the minor groove of double-helical DNA. A thermodynamic characterization of the DNA-binding properties exhibited by a six-ring hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (where Im = imidazole, Py = pyrrole, gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide), reveals an approximately 1-2 kcal/mol greater affinity for the designated match site, 5'-TGTTA-3', relative to the single base pair mismatch sites, 5'-TGGTA-3' and 5'-TATTA-3'. The enthalpy and entropy data at 20 degrees C reveal this sequence specificity to be entirely enthalpic in origin. Correlations between the thermodynamic driving forces underlying the sequence specificity exhibited by ImPyPy-gamma-PyPyPy-beta-Dp and the structural properties of the heterodimeric complex of PyPyPy and ImPyPy bound to the minor groove of DNA provide insight into the molecular forces that govern the affinity and specificity of pyrrole-imidazole polyamides.

Influence of cisplatin intrastrand crosslinking on the conformation, thermal stability, and energetics of a 20-mer DNA duplex.

cis-Diamminedichloroplatinum(II) (cisplatin) is a widely used anticancer drug that binds to and crosslinks DNA. The major DNA adduct of the drug results from coordination of two adjacent guanine bases to platinum to form the intrastrand crosslink cis-[Pt(NH3)2[d(GpG)-N7(1), -N7(2)]] (cis-Pt-GG). In the present study, spectroscopic and calorimetric techniques were employed to characterize the influence of this crosslink on the conformation, thermal stability, and energetics of a site-specifically platinated 20-mer DNA duplex. CD spectroscopic and thermal denaturation data revealed that the crosslink alters the structure of the host duplex, consistent with a shift from a B-like to an A-like conformation; lowers its thermal stability by approximately 9 degrees C; and reduces its thermodynamic stability by 6.3 kcal/mol at 25 degrees C, most of which is enthalpic in origin; but it does not alter the two-state melting behavior exhibited by the parent, unmodified duplex, despite the significant crosslink-induced changes noted above. The energetic consequences of the cis-Pt-GG crosslink are discussed in relation to the structural perturbations it induces in DNA and to how these crosslink-induced perturbations might modulate protein binding.

Characterizing the DNA binding modes of a topoisomerase I-poisoning terbenzimidazole: evidence for both intercalative and minor groove binding properties.

We have used a broad range of spectroscopic and viscometric techniques to demonstrate that the complexation of a cytotoxic, topoisomerase I-poisoning terbenzimidazole (5PTB) with the poly(dA).poly(dT) duplex exhibits properties characteristic of both intercalation and minor groove binding. Our results reveal the following features: (i) Optical melting profiles reveal that 5PTB binding enhances the thermal stability of the poly(dA).poly(dT) duplex; (ii) Fluorescence-detected 5PTB binding to the poly(dA).poly(dT) duplex reveals four apparent "site sizes," ranging from 1 to 13 base pairs (bp) per bound drug; (iii) Flow linear dichroism data suggest conformational heterogeneity among the poly(dA).poly(dT)-bound 5PTB molecules, with substantial contributions from drug molecules bound in the minor groove; (iv) Fluorescence resonance energy transfer data reveal properties characteristic of a significant contribution from an intercalative mode of binding; (v) Viscometric, fluorescence quenching, and netropsin competition data are consistent with 5PTB binding to poly(dA).poly(dT) by "mixed" modes, which are operationally defined as single or multiple binding populations that individually and/or collectively express both intercalative and minor groove binding properties. We comment on a potential correlation between drugs that exhibit such "mixed" mode binding motifs and those that express antineoplastic activity through inhibition of topoisomerase I.